Systems, Methods, and Apparatus for Single Molecule Sequencing

ABSTRACT

An embodiment generally relates to a system for analysis of an analyte. The system can include a transparent substrate. The system also includes an excitation light source configured to induce an evanescent wave excitation of a fluorescently labeled molecule near the access to the transparent substrate and a detector for detecting the fluorescently labeled molecule.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/800,440 entitled “Systems, Methods, And Apparatus For SingleMolecule Sequencing” filed on May 16, 2006 and is hereby incorporated byreference for all purposes.

FIELD

This invention relates generally to single molecule sequencing, moreparticularly, to detecting single molecules without the use of zero-modewaveguides.

DESCRIPTION OF THE RELATED ART

It is generally significant to reduce the background in assay systemsfor the detection of single molecule events. The background can comefrom the high concentration of labeled nucleotides in the detectionvolume. U.S. Patent Application 20030044781 ('781 Application), which ishereby incorporated by reference in its entirety, describes a method ofsequencing target nucleic acids having a plurality of bases. The '781Application describes the use of a zero-mode waveguide, a waveguide thatis approximately ten times smaller than the selected wavelength, topreclude propagation of light into a fluid, and consequently limit theexcited volume.

SUMMARY

An embodiment generally pertains to a system for analysis of an analyte.The system can include a transparent substrate. The system can alsoinclude an excitation light source configured to energize afluorescently labeled molecule by an inorganic absorber and a detectorconfigured to detect the fluorescently labeled molecule.

Another embodiment relates generally to a system for analysis ofanalyte. The system can include a transparent substrate coated with ametal layer and an exclusion coating deposited on the metal layer. Thesystem can further include a fluorescent labeled molecule configured todiffuse into holes of the exclusion coating and an excitation lightsource configured to produce plasmon-coupled emission excitation at asubstrate side of the exclusion coating.

Yet another embodiment pertains generally to a luminescence detectionsystem. The system includes a resonant optical cavity where luminophoreemission is elicited by evanescent excitation established at theboundary of a resonant optical cavity.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features of the embodiments can be more fully appreciated, asthe same become better understood with reference to the followingdetailed description of the embodiments when considered in connectionwith the accompanying figures, in which:

FIG. 1A illustrates a side view of an excitation device in accordancewith an embodiment;

FIG. 1B illustrates a more detailed view of an excitation zone;

FIG. 2 illustrates a top view of a section of the excitation deviceshown in FIG. 1;

FIG. 3A illustrates a side view of another excitation device inaccordance with an embodiment;

FIG. 3B illustrates a more detailed view of an excitation zone in theexcitation device shown in FIG. 3A;

FIG. 4 illustrates a top view of the excitation device shown in FIG. 3Ain accordance with yet another embodiment;

FIG. 5A illustrates a quantum excitation device in accordance with yetanother embodiment;

FIG. 5B illustrates a more detailed view of an excitation zone in theexcitation device shown in FIG. 5A;

FIG. 6A depicts an energy transfer zone around a quantum dot;

FIG. 6B illustrates an exemplary embodiment of multiple quantum dots;

FIG. 7 depicts a comparison of Rhodamine 6G and quantum dyes by emissionand excitation profiles;

FIG. 8 depicts emission profiles for some commercially available quantumdots;

FIG. 9 depicts the process for a tri-functional reagent for surfaceattachment to quantum dots;

FIG. 10 depicts the process of synthesis of quantum dots containingsingle polymerase, single biotin;

FIG. 11A depicts a Kretschmann configuration in accordance with yetanother embodiment;

FIG. 11B illustrates another SPCE system in accordance with yet anotherembodiment;

FIG. 11C illustrates yet another SPCE system in accordance with yetanother embodiment;

FIG. 11D illustrates a reverse Kretschmann system in accordance with yetanother embodiment;

FIG. 11E illustrates exemplary reflective and catadioptric lenses;

FIG. 11F illustrates yet another exemplary SPCE system in accordancewith yet another embodiment;

FIG. 12 depicts a conventional resonant laser cavity;

FIG. 13 illustrates a system in accordance with an embodiment of theinvention;

FIG. 14 illustrates a system in accordance with an embodiment of theinvention;

FIGS. 15A-D illustrate various embodiments of resonator cavityexcitation devices;

FIGS. 16A-B illustrate angles of light beams in various embodiments ofthe resonator cavity excitation device;

FIG. 17 illustrates a system in accordance with yet another embodiment;and

FIG. 18 illustrates a system in accordance with yet another embodiment.

It should be readily apparent to those of ordinary skill in the art thatthe figures depicted herein represent a generalized schematicillustration and that other components may be added or existingcomponents may be removed or modified.

DETAILED DESCRIPTION OF EMBODIMENTS

For simplicity and illustrative purposes, the principles of the presentinvention are described by referring mainly to exemplary embodimentsthereof. However, one of ordinary skill in the art would readilyrecognize that the same principles are equally applicable to, and can beimplemented in, all types of assay systems, and that any such variationsdo not depart from the true spirit and scope of the present invention.Moreover, in the following detailed description, references are made tothe accompanying figures, which illustrate specific embodiments.Electrical, mechanical, logical and structural changes may be made tothe embodiments without departing from the spirit and scope of thepresent invention. The following detailed description is, therefore, notto be taken in a limiting sense and the scope of the present inventionis defined by the appended claims and their equivalents.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all sub-ranges subsumedtherein. For example, a range of “less than 10” can include any and allsub-ranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all sub-ranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5.

One embodiment generally relates to systems and methods for detectingsingle molecule events. More specifically, an excitation device usinginternal reflection to prevent light from entering a fluid. Theexcitation device can include a substrate and a coating. The substratecan be a transparent material such as fused silica, glass, and orcrystalline materials. A coating can be applied to the substrate. Thecoating can be transparent, semi-opaque or opaque and should have alower index of refraction than the substrate. Microscopic holes can beimplemented within the coating. An analyte fluid can be placed on theexcitation device. An excitation light source, e.g., a laser at aselected frequency, is directed into the substrate and reflects off thecoating and fluid in the microscopic holes by total internal reflection(TIR). This reflection creates an evanescent wave extending into thecoating and its respective holes. The intensity of this evanescent wavequickly decays from the substrate surface. In some instances, thecoating can be referred to as an exclusion coating since molecules areexcluded from the excitation light except in the desired locations,typically, the microscopic holes.

Accordingly, this evanescent wave excitation can only illuminate theanalyte fluid that is close to the substrate of the excitation device.Because the evanescent wave intensity quickly decays from the substratesurface the coating can form a barrier that impedes the analyte fluid,i.e. dye molecules, from reaching the excitation except at the bottom ofthe holes where the analyte fluid is close to the substrate. Moreover,if the coating is transparent, light may be collected from above, belowor both sides of the substrate surface. The substrate surface in thedetection area is preferentially a plane but could be non-planar ifadvantageous for imaging. An example of a non-planar substrate surfacecan be a substrate implemented with multiple layers, where the layerscan be different materials with respective and appropriate indices ofrefraction.

Other embodiments pertain generally to a method of creating an energytransfer zone to locate the “useful” excitation zone to a smaller zonein proximity to an excitation light converter, where the excitationlight converter is chemically attached to the substrate. Morespecifically, embodiments may include at least one quantum dot as anexcitation light converter that absorbs light and transfers it to thelabeled moiety of interest. The transfer of energy can involve awavelength shift. The labeled moiety would then typically emit at alonger wavelength. In other embodiments, quantum dots of differing sizecan be used for different wavelengths.

In other embodiments involving two-photon excitation, the light can emitat a shorter wavelength than the excitation wavelength. This istypically done by using pulsed laser excitation to assure that twophotons can excite the fluorescent label in short time. Since theemission light is at a shorter wavelength than the excitation, it iseasier to reject the background by filtering, as the background istypically at a longer wavelength than the excitation. The wavelength offluorescence of photons emitted by dye molecules is typically a longerwavelength than the wavelength of the excitation photons that elicitedthe fluorescent emission.

Two-photon excitation may utilize either standard fluorophores, or mayutilize up-converting phosphors. An up-converting phosphor is made ofmixed-metal oxide nano-powders, including rare earth oxides. Y₂O₃:Yb,Er,Y₂O₂S:Yb,Er, Y₂O₂S:Yb,Ho, and Y₂O₂S:Yb,Tm, lanthanide compounds, areexamples from the literature. Unlike quantum dots or standardfluorophores, the emission light is a shorter wavelength than that ofthe excitation light, and thus has the advantages previously describedfor two-photon fluorophore excitation, while retaining the narrowemission bands and photo stability of quantum dots. Up-convertingphosphors can be made in various sizes, including powders as small as 20nm. As such, they can be used in manner similar to that of quantum dots,and thus any place where the term quantum dot is used hereafter, it mayalso refer to an up-converting phosphor, except where a quantum dot isdescribed.

The energy transfer for two-photon excitation can occur non-radiativelyvia an induced dipole-dipole interaction. Alternatively, the energytransfer can occur by radiative coupling. However, radiative couplingcan result in a larger excitation zone since radiative coupling drops by1/R² vs. 1/R⁶ for dipole-dipole interaction. Moreover, radiativecoupling can emit light that needs to be rejected or screened. Thisshift in the emission wavelength allows filters to block both excitationsources during detection. The blocking of the filters can be removedduring part of the process to allow target position determination.

Quantum dots can be made to provide a desired second excitation sincequantum dots produce a wavelength dependent on the size of the dot.Moreover, quantum dots can have a narrower spectral emission, shorterspectral tails and a wider absorption band than FRET dyes.

FIG. 1A illustrates a side view of an excitation device 100 inaccordance with an embodiment. As shown in FIG. 1, the excitation device100 can include a substrate 105 and an exclusion coating 110. In someembodiments, the substrate 105 and the exclusion coating 110 can beimplemented with material having low fluorescence characteristics tominimize background noise. In other embodiments, the substrate 105 canbe further specified to be fused silica (n=1.47), crystal quartz(n=1.543) or other type of low fluorescent glass, plastic orcombinations thereof.

In yet other embodiments, the substrate 105 can be implemented with highindex of refraction materials such as Ta₂O₅ (n=2.1) or TiO₂ (n=2.5-2.9).The use of high index materials in the substrate 105 can facilitate alight guiding layer for excitation light propagation. The light can becoupled into the light guiding layer using a prism or grating, asdescribed later with respect to FIG. 3AB. The exclusion coating 105 canbe deposited on this light guiding layer. Moreover, high-index ofrefraction substrates can allow more exclusion coating 110 options asthe coating and fluid index must be less than the substrate. In variousother embodiments, the evanescent wave can drop faster to reduce therequired exclusion coating thickness because a rapid change in the indexof refraction for material reduces the evanescent wave penetration depth140.

The implementation of the exclusion coating 110 can be dependent on theindex of the substrate 105 or light guiding layer 330. Moreparticularly, for high index substrates, many lower index materials maybe selected. For example, PMMA (n=1.491), polycarbonate (n=1.586),cyclic polyolefin (n=1.525) and other similar materials can be selectedfor the exclusion coating. In some embodiments, optical exclusioncoatings such as MgF₂ or glasses such as SiO₂ having an index less thanthe substrate 105 can also be used. In yet other embodiments, theexclusion coating 110 can be implemented using fused silica (n=1.47),Teflon FEP (n=1.341-1.347), Teflon AF (n=1.32), MgF₂ (n=1.38) may beused.

The exclusion coating 110 can be transparent or opaque. The exclusioncoating 110 can also be partially absorbing, reflective, or selectivelyreflective. For embodiments with opaque exclusion coatings and othertypes of exclusion coatings, unwanted light from the sample outside ahole is attenuated. Unwanted light can occur if the exclusion coating110 or substrate 105 is not properly formed resulting in leakage ofexcitation light 135 too far into the analyte fluid and reaching sample120. For embodiments with transparent exclusion coatings, emissions canbe collected from the side that interfaces with the analyte fluid sample120, the substrate side or both sides simultaneously. This is alsopossible with multi-layer structures.

Holes (or slots, channels, wells, etc.) 115 can be formed within theexclusion coating 110 to allow the analyte fluid sample 120 to interfacewith the substrate 105. In some embodiments, the analyte fluid sample120 can contain a dye labeled nucleotide with a non-fluorescent quencher125 on the triphosphate, i.e., dNTP. Placing a non-fluorescent quencher125 on the triphosphate quenches the free nucleotides. When incorporatedinto the DNA strand, the quencher is cleaved and the fluorescent dyelights up where all other free nucleotides are dim. Exclusion coating110 can be configured to exclude molecules from the excitation lightexcept in desired locations defined by the hole 115 or the pattern ofholes 115. Thus, the excitation light can only efficiently excitefluorescent molecules at the bottom of a hole 115.

For certain embodiments, the volume of the hole 115 can be small enoughto control background noise from diffusion events. For example,fluorescently labeled nucleotides at standard concentrations can beaccomplished with holes 115 sized between 30-150 nm or larger indiameter. Lower concentrations of labeled dNTPs can enable the user oflarger holes.

Although the holes 115 are depicted in side view in FIG. 1A, it shouldbe readily obvious to those skilled in the art that the shape of theholes 115 may be circular, rectangular, elliptical, or any other threedimensional shape. In some other embodiments, the holes 115 may havenon-vertical sides such as tapered or curved sides. For exclusion layerimplementations using multi-layer structures, the holes 115 can beconfigured by creating a larger hole, reapplying another exclusion layerand thus creating small holes. Other methods of creating holes 115 areknown to those skilled in the art.

The excitation device 100 can be in contact with the analyte fluidsample 120. The analyte fluid sample 120 can contain dyes, nucleotides,and other materials (e.g., dye labeled nucleotide non-fluorescentquencher on a triphosphate 125). During excitation, the light source 130can provide excitation light 135 which is directed into substrate 105 sothat total internal reflection TIR occurs at the substrate 105/coating110 or substrate 105/fluid 120 interface. When the light 135 isinternally reflected, a thin evanescent wave 140 is created in the lowerindex layer, i.e., in the exclusion coating 110.

FIG. 1B depicts a cross-section of one hole in the excitation zone 145in accordance with another embodiment. As shown, in FIG. 1B, theexcitation wave 140 is created from the TIR of the excitation light 135.The intensity of the evanescent wave 140 drops exponentially towardszero away from the surface so region 145 represents a zone ofsignificant energy near the substrate 105 at the bottom of the hole 115.TIR can also be used to reflect the light off the non-sample side of thesubstrate to reuse the excitation light 135. The evanescent wave 140 cancause molecules in the analyte fluid sample 120 that are near thesubstrate to fluoresce, e.g., molecule 150. The small excitation zone145 is the area that is both efficiently excited by the evanescent wave140 and in fluidic contact with the sample 120.

FIG. 2 illustrates a top view (i.e., fluid side view) of a section ofthe excitation device 100 shown in FIG. 1A. As shown in FIG. 2, theexclusion coating 110 can cover the substrate (not shown) except for theholes 115. Fluorescent molecules that migrate into the bottom of thehole 115 can enter the excitation zone 145 (see FIG. 1B) and emitfluorescent light 115A.

FIG. 3A illustrates a side view of an excitation device 300 inaccordance with an embodiment. As shown in FIG. 3A, the excitationdevice 300 is similar to excitation device 100. The excitation device300 can include a substrate 305 and an exclusion coating 310 with holes315. The excitation device 300 may be in contact with an analyte fluidsample 320, which can include fluorescently labeled nucleotide with anon-fluorescent quencher on the triphosphates 325. Substrate 305,exclusion coating 310, holes 315, and analyte fluid sample 320 havesimilar or identical properties/characteristics to the respectivecomponents of the excitation device 100.

The excitation device 300 can include a light guide layer 330. The lightguiding layer 330 can be implemented using high-index of refractionmaterials as previously described. Light 340 from a light source 335 canbe coupled into the light guiding layer 330 though a prism or grating asdescribed in U.S. Pat. No. 5,882,472, which is hereby incorporated byreference in its entirety.

During excitation, the light source 335 can provide light 340 which isdirected into the light guiding layer 330 so that total internalreflection TIR occurs at the light guiding layer 330/exclusion coating310 and light guiding layer 330/fluid 320 interfaces. When light isinternally reflected, a thin evanescent wave 345 is created in the lowerindex of refraction layer, i.e., in the exclusion coating 310 and in thefluid. The evanescent wave can cause molecules in the analyte fluidsample 320 that are near the light guiding layer 330 to fluoresce, e.g.,molecule 355.

FIG. 3B depicts a cross-section of one hole in the evanescent wave 345in accordance with another embodiment. As shown, in FIG. 3B, theevanescent wave 345 is created from the TIR of the excitation light 340in the light guiding layer 330. The intensity of the evanescent wave 345drops exponentially towards zero moving away from the boundary so region350 represents a zone of significant energy near the light guiding layer330 at the bottom of hole 315. TIR can also be used to reflect the lightoff the non-sample side of the light guiding layer 330 to reuse theexcitation light 340. The evanescent wave 345 can cause molecules in theanalyte fluid sample 320 that are near the light guiding layer 330 tofluoresce, e.g., molecule 355. The small excitation zone 350 is theareas that are both excited by the evanescent wave 345 and in fluidiccontact with the sample 320.

FIG. 4 illustrates a top view of the excitation device 300 shown in FIG.3A in accordance with yet another embodiment. As shown in FIG. 4, theexclusion coating 310 can cover the light guiding layer 330 (not shown)except for the holes 315. Fluorescent molecules that migrate to thebottom of the hole 315 can enter the excitation zone 350 (see FIG. 3B)and emit fluorescent light 315A.

FIG. 5A illustrates a quantum excitation device 500 in accordance withyet another embodiment. As shown in FIG. 5A, the quantum excitationdevice 500 is similar to excitation device 100. The quantum excitationdevice 500 can include a substrate 505 and an exclusion coating 510 withholes 515. The substrate 505 and the exclusion coating 510 can beimplemented with the materials as previously described with respect toFIGS. 1A-4. Moreover, the index of refraction of the exclusion coating510 is less than the substrate 505.

The quantum excitation device 500 can be in contact with an analytefluid sample 520, which can also include fluorescently labelednucleotide with a non fluorescent quencher on the triphosphates 525.Substrate 505, exclusion coating 510, holes 515, and analyte fluidsample 520 have similar and/or identical properties/characteristics tothe respective components of the excitation device 100. The quantumexcitation device 500 can also include a light source 530. Light 535from the light source 530 can be coupled into the substrate 505. Similarto excitation device 300 (see FIG. 3AB), excitation device 500 can beimplemented with a light-guiding layer similar to the light-guidinglayer 330 in other embodiments.

Quantum excitation device 500 can include at least one quantum dot 540.A quantum dot can be a semiconductor nanostructure that confines themotion of conduction band electrons, valence band holes or excitons(bound pairs of conduction band electrons and valence band holes) in allthree spatial directions. Quantum dots can include those made of siliconand germanium, and presumably of other materials, as well as CdSe.Quantum dots can also be fabricated as core-shell structures with, e.g.,CdSe in the core and ZnS in the shell. It should be readily obvious thatother types of materials could be used to create quantum dots and fallwithin the spirit and scope of the claimed invention.

Other embodiments can use multiple quantum dots of differing size and/ortypes. During the excitation phase, the quantum dot 540 can create anenergy transfer zone. More particularly, the light source 530 canprovide light 535 which is directed into the substrate 505 so that TIRoccurs at the substrate 505/exclusion coating 510 and at the substrate505/fluid 520 interface. When the light 535 is internally reflected, athin evanescent wave 545 is created in the lower index of refractionlayer, i.e., in the exclusion coating 510. The evanescent wave 545 cancreate a small excitation zone 550 (see FIG. 5B) due to the rapid decayof the evanescent wave 545. The quantum dot 540 can absorb the energy inthe small excitation zone 550 which converts the excitation energy intothe desired wavelength. The conversion process can create an energytransfer zone surrounding the quantum dot 540, which is depicted in FIG.6A.

As shown in FIG. 6A, the energy in the transfer zone 605 can betransferred to a labeled moiety, that is, an excited dye-labelednucleotide 610. The dye-labeled nucleotide 610 can be attached to thequantum dot 540 by an enzyme 615. In some embodiments, multiple quantumdots of differing types and/or sizes can be used to have severalexcitation wavelengths, which are illustrated in FIG. 6B.

As shown in FIG. 6B, quantum dot one (labeled as “Q1”) 650 may create asurrounding energy transfer zone 655. Quantum dot two (labeled as “Q2”)660 may also create a surrounding energy transfer zone 665. The energyzones 655, 665 may be created by the presence of excitation energy.Since the quantum dots, 650 and 660, are of differing size, and may beof a differing type, they can both be used to generate severalwavelengths. Like the embodiment shown in FIG. 6A, a dye labelednucleotide 610 can be attached to the quantum dots 650, 660 by at leastone enzyme 615.

Returning to FIG. 5A, the quantum excitation device also includes adetector 550, which is configured to identify the type of emission fromthe quantum dots. The detected emission is then directed to a computer(not shown) where the molecule corresponding to the emission isidentified and its identity stored.

FIG. 7 depicts a comparison 700 of (a) the excitation and (b) emissionprofiles between CdSe quantum dots and Rhodamine 6G dye. The quantumdots emission spectrum (in (b)) is nearly symmetric and narrower in peakwidth. However, its excitation profile is broad and continuous. Thus,the quantum dots can be efficiently excited by wavelengths shorter than530 nm. By contrast, the organic dye Rhodamine 6G has excitation peakswhich limit excitation wavelength choices.

FIG. 8 depicts emission profiles 800 for some commercially availablequantum dots. As shown in FIG. 8, the wavelength spread fromcommercially available samples can be considered reasonable.Accordingly, it is anticipated that careful selection and/or fabricationcan enable even tighter distributions.

Returning to FIG. 6A, quantum dot 540 is depicted attached to the dyelabeled nucleotide 610 and enzyme 615. Ideally, a single enzyme 615 isattached to a single quantum dot 540 and the attached pair is thenattached to the substrate 505. In the quantum excitation device 500, itis further desired to have a single moiety in each excitation zone. Theodds of getting one and only one of each moiety and attached pair in awell are low if standard Poisson distributions are created. At best, itwould by 0.37² (14 percent) but typically something in the 10 percent ismore likely. This low useful density can decrease the effectivethroughput of a system.

One conventional strategy is to enrich the number of correct pairs. Forexample, one solution is to perform attachment chemistry using dilutesolutions such that most molecules are not part of a pair. Because ofthe low concentrations, there are few moieties with more than one ofeach. This solution can then be enriched by selection (such as using aFluorescent Activated Cell Sort instrument) or by pullout using achemical hook such as Streptavidin-Biotin, which is known to thoseskilled in the art.

Embodiments of the quantum excitation device can use a linker moleculethat has three attachment sites. FIG. 9 depicts the process 900 for atri-functional reagent for surface attachment to quantum dots 540. FIG.10 depicts the process 1000 of synthesis of quantum dots 540 containingsingle polymerase, single biotin.

Through the processes of FIGS. 9-10, an enzyme containing at least oneCystine can be attached to each maleimide containing quantum dot bydriving the reaction to completion. Thus, each quantum dot that has asingle hook biotin attached will have a single enzyme. This can beattached to the substrate 505. In other embodiments, the well caninclude multiple streptavidins, as described in U.S. ProvisionalApplication 60/689,692, filed on Jun. 10, 2005, entitled “Method andSystem for Multiplex Genetic Analysis,” which is hereby incorporated byreference in its entirety.

Accordingly, embodiments of the quantum excitation device can providesome advantages over the conventional systems. For example, the quantumdots in the excitation device can provide multiple methods ofattenuation in a single system. Another advantage is that evanescentlayer of excitation is approximately 100 nm thick using TIR. Thisreduces the excitation zone by limiting excitation light in the Z-axis.Moreover, the excitation source can be at a wavelength that weaklyexcites the moiety of interest. For example, a wavelength of 405 nmwould weakly excite the Rhodamine 6G-dye shown in FIG. 7, but thequantum dots are well excited at these shorter wavelengths. This wideabsorption range also permits the use of different excitation sources.For example, 405 nm lasers (used in high definition DVD players) aremuch cheaper than a 488 nm laser used in conventional fluorescentexcitation.

Yet another advantage of the embodiments is that quantum dots or otherlong life donors create a second excitation zone. This limits the numberof sources in the all directions. Incorporated or bound nucleotides seehigh levels of efficiently absorbed light only if they are very near thequantum dot. The effective energy transfer distances are on the order of5 nm so this creates small scale excitation zones.

Yet another advantage is free nucleotides that diffuse into the energytransfer zone (see 605 in FIG. 6A) will typically exit very rapidly dueto the extremely small excitation volume. The time for free nucleotidesto diffuse in and out of the energy transfer zone is much shorter thanthat for an enzyme to incorporate a nucleotide. This improves moietydetermination based on accumulated signal (for example via CCDdetection) as opposed to a single photon counting.

Another embodiment using two-photon excitation can be used to reducebackground noise. With two-photon excitation, the emission of light isat shorter wavelength than the excitation light. Two-photon emission isonly generated where a high excitation level exists. Any backgroundfluorescence is at longer wavelength than the excitation source and iseasily filtered out using filters such as short pass or band-passfilters.

Another embodiment can use an alternative to TIR. These embodiments usesurface plasmon-coupled direction emission (“SPCE”). SPCE can includeusing angular emission profiles of dye molecules excited by surfaceplasmon waves at a metal surface, which is described in WO 2005/003743.The entirety of WO 2005/003743 is incorporated by reference. SPCEtechniques can be combined with the embodiments of the excitation devicedescribed previously to provide improved excitation and collection withzeptoliter excitation volumes, which are depicted in FIG. 11A-D.

FIG. 11A illustrates an exemplary embodiment of a Kretschmannconfiguration system 1100A. In this configuration, energy from thefluorescent molecule is emitted as light on the other side of the metalsurface. Background can be suppressed and both excitation and emissionefficiencies can be significantly improved. If the fluorescent moleculeis too close to the metal surface (<20 nm) than the energy is quenched.A spacer layer can be used to prevent such intimate contact. The spacerlayer can be dielectric material with a low fluorescence such as SiO₂.

SPCE techniques can offer some advantages over TIR systems. For example,the choices for the exclusion coating are larger because the evanescentwave is not created by the transition to a low index material. Inaddition, the directional nature of the emitted light can simplify thecollection optics and improve background rejection. Finally, theproximity to metal has been shown to increase photo stability, which isespecially important for single molecule applications.

FIG. 11B illustrates another embodiment of a SPCE system 1100B. As shownin FIG. 11B, an excitation device can include a substrate 1110Binterfaced with a metal layer 1115B, and an exclusion coating 1120B. Thesubstrate 1110B and the exclusion coating 1120B can be implemented withmaterials and techniques as described earlier. The metal layer 1115B canbe implemented with gold, silver or other metals appropriate for SPCEwith a thickness of 5-75 nm or more, preferably 25-30 nm for gold, and50 nm for silver. In addition, another metal such as chromium may beapplied to the substrate in order to improve adhesion while notinterfering with the refractive index of the thicker layer which issubsequently applied; this layer has been reported to be between 2 to 5nm. In some embodiments, the metal layer 1115B can be a non-metallicmaterial that has a significant change in index from the substrate1110B.

The exclusion coating 1120B can be deposited or grown on one side of themetal layer 1115B. The exclusion coating 1120B can be implemented withhigh-index of refraction material (e.g., fused silica, polycarbonate) aspreviously described with respect to FIGS. 1A-4. Holes (channels, slots,etc.) 1125B can be formed in the exclusion coating 1120B for the analytesolution 1130B to contact with the metal layer 1115B. Like some otherembodiments, dye molecules (e.g., labeled nucleotides) can migrate tothe bottom of the hole 1125B.

The SPCE system 1100B can also include a lens 1135B implemented as atruncated sphere, hemisphere or other similar three dimensional shape.The lens 1135B can be configured to be in an aplanatic condition, thatis, the lens 1135B doesn't create spherical aberrations or coma. Thelens 1135B can be configured to interface with the substrate 1110Bthrough an index matching fluid 1140B which can couple light in and outat the steep angles required. In some embodiments, the excitation source1145B can operate at a single frequency or, in the case of thisembodiment, multiple frequencies such as 488 nm and 532 nm.

Accordingly, when the light from the excitation sources 1145B hits thesubstrate 1110B, a surface plasmon is excited at the interface of themetal layer 1115B and the substrate 1110B. The surface plasmon can beconsidered as a ray of light bound onto a surface of the metal layer1115B propagating along the surface and presenting itself as an electricfield. The electric field extends into the exclusion layer 1120B and theholes 1125B and rapidly decays with distance from the metal surface.Prior to the decay of the electric field, an energy transfer zone can becreated similar to the zone of significant energy 145 of FIG. 1B. Thedye molecules (e.g., labeled nucleotides) that have migrated into thisenergy transfer zone can be energized and fluoresce and much of theenergy can be emitted by plasmon coupled emission which forms a cone ofemission light 1150B.

In some embodiments of 1100B, the exclusion coating 1120B can beimplemented with material that does not support total internalreflection, i.e a equal or higher index than the substrate, i.e., notmatching with index matching fluid 1140B. The coating can be lower butdoesn't have to be because TIR does not occur. In other embodiments, theexclusion coating 1120B can be implemented with materials with a lesserindex of refraction. The material can be a metal or a non-transparentmaterial as known to those skilled in the art

FIG. 11C illustrates another exemplary SPCE system 1100C in accordancewith yet another embodiment. As shown in FIG. 11C, the system 1100Cincludes an excitation device. The excitation device can include asubstrate 1110C, which can be implemented with similar materials asdescribed earlier with respect to FIG. 1A and FIG. 3A. The substrate1110C can be in contact with a metal layer 1115C. The metal layer 1115Ccan be implemented with materials similar to the metal layer 1115B ofFIG. 11B as described herein above. In some embodiments, the metal layer1115C can be a non-metallic material that has a significant change inindex from the substrate 1110C. The metal layer 1115C and substrate1110C interface can be used for SPCE excitation.

A spacer layer 1120C can be formed on one side of the metal layer 1115C.The spacer layer 1120C can be implemented with SiO₂ in a range of 1-100nm and preferably 5-25 nm thick. A metal coating 1125C can be formed onone side of the spacer layer 1120C. The metal coating 1125C can beimplemented with material such as gold, aluminum, titanium or othersimilar materials. Holes 1130C can be formed within the metal coating1125C, where dye molecules (e.g., labeled nucleotides) 1135C in a fluid1140C can migrate to the bottom of the holes 1130C.

Accordingly, when the light from an excitation source substrate 1110C, asurface plasmon is excited at the interface of the metal layer 1115C andthe substrate 1110C. The surface plasmon can create an electromagneticfield that extends into the holes 1130C that rapidly decays. An energytransfer zone can be created similar to the zone of significant energy145 of FIG. 1B. The labeled nucleotides 1135C that have migrated intothis energy transfer zone can be energized and fluoresce.

Moreover, a zone of quenching 1145C can be created due to theinteraction of the dyes with the thick metal coating 1125C. The zone ofquenching 1145C can be approximately 20 nm. Any dye molecules in thiszone are suppressed from fluorescing.

FIG. 11D illustrates a reverse Kretschmann excitation system 1100D forSPCE. As background, reverse Kretschmann (“RK”) is a conventionalapproach for SPCE. In RK, the excitation is from the sample side but theemission is on the other side of the metal surface. When excitationlight is brought in on the sample side, it excites dye molecules alongthe way. There are many dye molecules away from the surface and some ofthe emitted light will transmit through the metal surface since themetal is not thick enough to block all the light. This will cause toohigh a background. Moreover, the excitation light can bleach the samplesbefore they get near the surface. The embodiment shown in FIG. 11D cansolve these issues.

As shown in FIG. 11D, the excitation system 1100D can include anexcitation device 1105D, which can include a substrate 1110D interfacedwith a metal layer 1115D, and a spacer layer 1120D. The substrate 1110D,metal layer 115D and spacer layer 1120D can be implemented withmaterials and techniques as described earlier with respect to FIGS.11A-C. In some embodiments, the metal layer 1115D can be a non-metallicmaterial that has a significant change in index from the substrate1110D. The spacer layer 1120D can be in contact with an analyte solution1125D. Quantum dots 1130D can be bound to the spacer layer 1120D. Forclarity, quantum dots are described but it is understood that otherabsorbers could be used such as noble metal nanodots (colloidalparticles).

One side of the substrate 1110D can be in contact with an index matchingfluid 1135D. A lens 1140D can encompass the index matching fluid 1135Don the substrate 1110D. The lens 1140D may be implemented as a truncatedsphere, hemisphere or other similar three-dimensional shape. The lens1140D can be configured to be in an aplanatic condition, that is, thelens 1140D avoids spherical aberrations. The lens 1140D may also beimplemented as a reflective or catadioptric lens in general, and as aSchwarzschild objective in particular. While the hole required throughthe primary mirror of centered reflective systems and the obscurationdue to the secondary minor are limitations for many imaging systems,they are not limitations for a Kretschmann system. They are notlimitations since the elicited luminescent is emitted into an annularpattern that is entirely collectible by a two minor system, even thoughthey comprise hole in the primary and an obscuration due to thesecondary. Exemplary reflective 1190D and catadoptric 1195D lenses areshown in FIG. 11E. The system 1100D can also include an aperture 1145D,which is configured to block primary excitation background emissions,and plasmon emissions from the quantum dot 1130D from collection sensors(not shown).

Light beam 1150D (e.g., wavelength 405 nm, or 980 nm for up-convertingphosphors) from an excitation source (not shown) can excite the quantumdots 1130D, which generate a small emission zone with a low background.Since the excitation light is spectrally far removed from the dyeemission and absorbance, the excitation light beam 1150D can easily beoptically filtered and the dyes poorly absorb the primary emissionlight, which reduces photo degradation of the dyes. The light emittedfrom the dyes near the colloidal particles 1130D can be emitted in anarrow angular distribution on the substrate 1110D side in a cone oflight 1155D. The cone of light 1155D can be efficiently collected andfiltered and then detected by a sensor such as a charge coupleddetector. Some of the excitation light beam 1160D can be transmitted tothe substrate side 1110D but this light can be easily blocked, filteredor directed away from the collection area.

FIG. 11G illustrates another exemplary SPCE system 1100F in accordancewith yet another embodiment. As shown in FIG. 11F, the system 1100Fincludes an excitation device. The excitation device can include asubstrate 1110F, which can be implemented with similar materials asdescribed earlier with respect to FIG. 1A and FIG. 3A. The substrate1110F can be in contact with a thin metal layer 1115F. The metal layer1115F can be implemented with materials similar to the metal layer 1115Bof FIG. 11B as described herein above and with a thickness of about25-50 nanometers. A second metal layer 1120F can deposited over themetal layer 1115F. The second metal layer 1120F can be a thickness ofabout 100 nm and implemented with materials similar to the metal layer1115F or a different metal. The thickness of the second metal layer1120F prevents coupling of any surface plasmons and reflects the surfaceplasmons. Accordingly, the metal layer 1115F and substrate 1110Finterface can be used for SPCE excitation. The first metal can be pureor an alloy provided plasmons are supported. The addition of the secondlayer should not support surface plasmons (i.e. it can be a thickerlayer of same metal or it can be a different metal or alloy.

Holes 1130F can be formed within the second metal layer 1120F, where dyemolecules (e.g., labeled nucleotides) 1135F in a fluid 1140F can migrateto the bottom of the holes 1130F. Holes can be any size where smallerholes allow higher concentrations for faster kinetics. Size can be lessthan 1000 nm preferably less than 200 nm and still more preferably lessthan 100 nm.

Accordingly, when the light from an excitation source substrate 1110F, asurface plasmon is excited at the interface of the metal layer 1115F andthe substrate 1110F. The surface plasmon can create an electric fieldthat extends into the holes 1130F that rapidly decays. The labelednucleotides 1135F that have migrated into this energy transfer zone canbe energized and fluoresce.

Moreover, a zone of quenching 1145F can be created due to theinteraction of the dyes with the second metal layer 1120F. The zone ofquenching 1145E can be approximately 20 nm. Any dye molecules in thiszone are suppressed from fluorescing.

Returning to FIGS. 1A and 3A, the respective light sources (130 and 335)can be generally described as sources of excitation energy at a selectedwavelength. In conventional systems, the light source may be a resonatorcavity laser as depicted in FIG. 12. As shown in FIG. 12, a resonatorcavity laser 1200 includes highly reflective minors 1205. Within thecavity 1210, a laser pump 1215, for example, an argon ion laser pump,may generate the selected wavelength, which is reflected between themirrors 1205 until it can pass through the output coupler 1220.

Conventional resonator cavity lasers have drawbacks and disadvantages.For example, the laser power outside the resonator cavity laser isapproximately 100 times less than inside the resonator cavity.Accordingly, additional embodiments generally pertain to increasingfluorescence generation. More particularly, embodiments can enhanceoptical processes by placing fluorophores in communication with theresonator cavity so that they are exposed to an excitation fieldamplitude comparable to that existing within the cavity rather than thatof a beam substantially outside the cavity. In other embodiments, anefficient use of laser power can be exciting fluorophores located inholes of the embodiments of the excitation devices implemented with anexclusion coatings or metallic coatings comprising holes with dimensionand pitch less than the wavelength of the laser on silica substrate byusing the excitation device 1320 as a cavity mirror, as depicted in FIG.13.

FIG. 13 illustrates a system 1300 in accordance with an embodiment ofthe invention. As shown in FIG. 13, the system 1300 can include highlyreflective minors 1305A and 1305B. A light source 1315 can be set upinside of a resonant cavity 1310. Positioned inside a laser resonantcavity can be the substrate 1320, e.g. Substrate 105 shown in FIG. 1.The substrate 105 should be configured to efficiently reflect the light,for example by TIR, at the exclusion layer interface such that theevanescent wave can excite the fluorophores that penetrate the holes inthe exclusion layer while the resonant beam circulates within thecavity. Technically speaking, the fluorophores are outside the cavitysince the TIR surface comprises one of the resonator boundaries. Butthere is an evanescent component of the intra-cavity beam that extendsnanometers beyond the interface.

FIG. 14 illustrates a system 1400 in accordance with an embodiment ofthe present invention. As shown in FIG. 14, system 1400 depictsexcitation of the luminophores on the excitation device 1420 through anexternal laser 1410. The system 1400 can include highly reflectiveminors 1405. The external laser 1410 may generate coherent light energyat the selected frequency directed into the resonant cavity defined byminors 1405. An active cavity alignment device 1414 establishes andmaintains the cavity length and alignment necessary for resonance.Resonance allows a high field to build up in the cavity. The substrateof the excitation device 1420 can be configured to efficiently interfacewith the resonant cavity such that the evanescent wave can excite thefluorescently labeled dNTPs that migrate into the holes in the exclusioncoating. The path of the coherent light energy can reflect to the secondhighly reflective mirror 1405B and then be reflected back towards theexcitation device 1420.

FIGS. 15A-D depict various embodiments of the resonator cavityexcitation devices 1500. As shown in FIGS. 15 A-C, a substrate 1505 canbe interfaced with an aqueous luminescent analyte 1510. The interfacecan constitute a TIR interface 1525. The resonator cavities of 1515 arebounded by the highly reflective mirrors 1520 and the TIR interface1525. Light path 1530 represents the various angles of light beam intothe substrates 1505. For the sake of clarity, the holes or wells havebeen omitted. It should be noted that wells may be omitted in the actualdevice and the devices depicted in FIGS. 15A-D would still function. Forinstance, one could pattern a hybridization array on the liquid side ofthe interface and use resonant cavity enhancement. In some embodiments,the substrate can mean at least one piece of refractive material. Forthe embodiments shown in FIGS. 15A-D, one piece of glass is depicted.Preferably, one would have a very thin layer of glass in optical contactwith the prism or lens and have the fluid in contact with the other sideof the thin layer.

FIG. 15A depicts a resonator cavity where the light beam enters theprism at Brewster's angle, which is depicted in greater detail in FIGS.16A-B. FIGS. 16A-B illustrates an exemplary resonator cavity excitationdevice 1600 showing the angles to initiate TIR. As shown in FIG. 16A,the cross-section of excitation device 1600 includes a single hole 1615.The substrate 1605 of the excitation device can be implemented withsilica (n=1.457) and the exclusion coating 1610 is implemented with MgF₂(n=1.377). Laser light can be any available color appropriate for thedyes used. For example, 488 nm or 632 nm are commonly available lasercolors. Multiple lasers can either be used independently orsimultaneously.

Within the substrate 1605, light path 1625 can approach at the criticalangle (Brewster's angle) 55.53 degrees to achieve zero-reflectionpassage from the air to the substrate 1605 The light path 1625approaches the hole 1615 at the critical angle or greater to induce TIRat the TIR interface 1620. For this embodiment, the critical angle is71.26 for 633 nm light although the angle of incidence can be larger.The reflected light can exit at the same angle but can deviate dependingon the angle of incidence, wavelength of the light, and/or materials.

As depicted in FIG. 16A, the angle of the side wall of the substrate1605 is 105 degrees from the interface between the substrate 1605 andexclusion coating 1610.

FIG. 16B depicts a resonator cavity excitation device 1600 where thelight path 1625 approaches the hole 1615 at the critical angle orgreater to induce TIR. For this embodiment, the critical angle is 72.578degrees from incidence. The reflected light can exit at the same anglebut can deviate depending on the angle of incidence, wavelength of thelight, and/or materials.

Turning to FIG. 15B, this figure depicts another embodiment of theresonant cavity excitation device where the highly reflective minors1520 of the resonator cavity direct light beam 1525 at normal incidence.Turning to FIG. 15C, this figure depicts yet another embodiment of theresonator cavity excitation device 1500. The substrate 1505 can beconfigured to be a hemisphere. The highly reflective minors 1520 arepositioned to let the light beam 1525 enter the substrate 1505 at normalincidence.

FIG. 15D depicts yet another embodiment of the resonator cavityexcitation device 1500. In this embodiment, multiple cavities 1550-1565can be formed. Each cavity can support a laser at a selected wavelength.Accordingly, for this embodiment, four different wavelengths can be usedin detection. It should be readily obvious to one of skilled in the artthat any number of cavities can be support without departing with thescope of the invention,

FIG. 17 illustrates a system 1700 in accordance with yet anotherembodiment. As shown in FIG. 17, an excitation device can includesubstrate 1705 implemented with a silica material and an exclusioncoating 1710 implemented with aluminum. A hole 1712 can be formed withinthe exclusion coating 1710 to interact with a dye solution 1714. Thesubstrate 1705 can be configured to have prism surfaces 1715AB. Thesystem 1700 can also include highly reflective mirrors 1720AB.

During excitation, an input laser can emit coherent light energy 1725 ata selected wavelength through the highly reflective minor 1720A throughan input coupler (not shown). The coherent light energy 1725 from thehighly reflective minor 1720A can be transmitted toward a side of thefixed prism 1715A. The coherent light energy 1725 can also be directedto highly reflective minor 1720B and reflected toward another side ofthe prism surface 1715B. The prism surface 1715B can be configured toreflect the light toward the hole 1712.

Yet another embodiment uses optical fibers and is depicted in FIG. 18.As shown in FIG. 18, system 1800 includes an excitation device 1805which launches light down one or more optical fibers 1810. A detectionarea 1815 may be created over a portion of the fibers 1810. A coating onthe fibers can designed to induce evanescent waves of the surface of thefiber. In the detection area 1815, small holes (not shown) may be placedin the coating such that the coating of the fibers 1810 acts as anexclusion coating. The run of optical fiber 1810 can include loops 1820large enough to preserve TIR.

While the invention has been described with reference to the exemplaryembodiments thereof, those skilled in the art will be able to makevarious modifications to the described embodiments without departingfrom the true spirit and scope. The terms and descriptions used hereinare set forth by way of illustration only and are not meant aslimitations. In particular, although the method has been described byexamples, the steps of the method may be performed in a different orderthan illustrated or simultaneously. Those skilled in the art willrecognize that these and other variations are possible within the spiritand scope as defined in the following claims and their equivalents.

1. A system for analysis of an analyte comprising: a substrate; a fluidsample configured to contact the substrate; an excitation light sourceconfigured to energize a fluorescently labeled molecule by an inorganicabsorber bound to the substrate; and a detector configured to detect thefluorescently labeled molecule.
 2. The system of claim 1, wherein theinorganic absorber is a quantum dot.
 3. The system of claim 1, whereinthe inorganic absorber is an upconverting phosphor.
 4. The system ofclaim 1, wherein the excitation light source induces the evanescent waveby internal reflection.
 5. The system of claim 1, wherein the substrateand the fluid sample each have different index of refraction from theother.
 6. The system of claim 1, wherein the inorganic absorber createsan energy transfer zone at a first wavelength.
 7. The system of claim 6,further comprising: a labeled moiety; a dye labeled nucleotide; and anenzyme, wherein one end of the enzyme is attached to the a labeledmoiety and a second end of the enzyme is attached to the dye labelednucleotide.
 8. The system of claim 7, further comprising a secondinorganic absorber, wherein the second inorganic absorber is configuredto create a second energy transfer zone at a second wavelength.
 9. Thesystem of claim 7, wherein the fluorescently labeled moiety includes afluorescent dye component, a quencher component and a nucleotidecomponent.
 10. The system of claim 9, wherein the fluorescent dyelabeled nucleotide by an end of the enzyme the fluorescent dye componentis configured to fluoresce upon cleavage from the quencher component andthe nucleotide component.
 11. A system for analysis of analytecomprising: a transparent substrate coated with a metal layer; anexclusion coating deposited on the metal layer; a fluorescently labeledmolecule configured to diffuse into holes in the exclusion coating; andan excitation light source configured to produce plasmon-coupledemission at a substrate side of the exclusion coating.
 12. The system ofclaim 11, wherein the exclusion coating is implemented with an index ofrefraction material higher than a material of the substrate.
 13. Thesystem of claim 11, wherein the plasmon coupled emission form an energytransfer zone in the holes in the exclusion coating.
 14. The system ofclaim 13, wherein a cone of emission light is formed in response thefluorescently labeled molecule entering the energy transfer zone.
 15. Aluminescence detection system, the system comprising a resonant opticalcavity, wherein luminophore emission is elicited by evanescentexcitation established at the boundary of the resonant optical cavity.16. The system of claim 15, wherein the resonant optical cavity furthercomprises: an excitation light source configured to produce light at aselected wavelength; a substrate; and a plurality of mirrors, whereinone mirror is position to reflect and deflect light from the excitationlight source to the substrate which reflects the light to the secondmirror, which reflects the light and redirects the light substantiallythe reverse direction as the incoming light.
 17. The system of claim 15,further comprising: an excitation light source configured to producelight at a selected wavelength; a total internal reflection interface;and a plurality of mirrors, wherein the resonant optical cavity isbounded by the plurality of mirrors and the total internal reflectioninterface.
 18. The system of claim 17, wherein the light from theexcitation light source enters the resonant optical cavity at theBrewster's angle to induce total internal reflection.
 19. The system ofclaim 17, wherein the light from the excitation light source enters theresonant optical cavity at normal incidence.
 20. The system of claim 15,further comprising: a plurality of light source, each source configuredto produce associated light at a selected wavelength; a plurality oftotal internal reflection interface; a plurality of mirrors furthercomprising of a plurality of subsets of mirrors, each subset of mirrorsassociated with a light source, wherein a plurality of resonant cavitiesare formed, each resonant cavity bounded by an associated subset ofmirrors and associated total internal reflection interface and suppliedwith light from an associated light source.